Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Jun 1997; 179(11): 3773–3782.
PMCID: PMC179177

Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants.


Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.

Full Text

The Full Text of this article is available as a PDF (976K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Achey PM, Wright CF. Inducible repair of thymine ring saturation damage in phi X174 DNA. Radiat Res. 1983 Mar;93(3):609–612. [PubMed]
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Basu AK, Loechler EL, Leadon SA, Essigmann JM. Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7677–7681. [PMC free article] [PubMed]
  • Bespalov IA, Purmal AA, Glackin MP, Wallace SS, Melamede RJ. Recombinant Phabs reactive with 7,8-dihydro-8-oxoguanine, a major oxidative DNA lesion. Biochemistry. 1996 Feb 20;35(7):2067–2078. [PubMed]
  • Boiteux S, Huisman O. Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12. Mol Gen Genet. 1989 Jan;215(2):300–305. [PubMed]
  • Boiteux S, O'Connor TR, Laval J. Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein. EMBO J. 1987 Oct;6(10):3177–3183. [PMC free article] [PubMed]
  • Breen AP, Murphy JA. Reactions of oxyl radicals with DNA. Free Radic Biol Med. 1995 Jun;18(6):1033–1077. [PubMed]
  • Cabrera M, Nghiem Y, Miller JH. mutM, a second mutator locus in Escherichia coli that generates G.C----T.A transversions. J Bacteriol. 1988 Nov;170(11):5405–5407. [PMC free article] [PubMed]
  • Castaing B, Boiteux S, Zelwer C. DNA containing a chemically reduced apurinic site is a high affinity ligand for the E. coli formamidopyrimidine-DNA glycosylase. Nucleic Acids Res. 1992 Feb 11;20(3):389–394. [PMC free article] [PubMed]
  • Chan E, Weiss B. Endonuclease IV of Escherichia coli is induced by paraquat. Proc Natl Acad Sci U S A. 1987 May;84(10):3189–3193. [PMC free article] [PubMed]
  • Chen H, Bjerknes M, Kumar R, Jay E. Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res. 1994 Nov 25;22(23):4953–4957. [PMC free article] [PubMed]
  • Chetsanga CJ, Lindahl T. Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli. Nucleic Acids Res. 1979 Aug 10;6(11):3673–3684. [PMC free article] [PubMed]
  • Clark JM, Beardsley GP. Thymine glycol lesions terminate chain elongation by DNA polymerase I in vitro. Nucleic Acids Res. 1986 Jan 24;14(2):737–749. [PMC free article] [PubMed]
  • Cunningham RP, Weiss B. Endonuclease III (nth) mutants of Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jan;82(2):474–478. [PMC free article] [PubMed]
  • Cunningham RP, Saporito SM, Spitzer SG, Weiss B. Endonuclease IV (nfo) mutant of Escherichia coli. J Bacteriol. 1986 Dec;168(3):1120–1127. [PMC free article] [PubMed]
  • Demple B, Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem. 1994;63:915–948. [PubMed]
  • Demple B, Linn S. DNA N-glycosylases and UV repair. Nature. 1980 Sep 18;287(5779):203–208. [PubMed]
  • Evans J, Maccabee M, Hatahet Z, Courcelle J, Bockrath R, Ide H, Wallace S. Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis. Mutat Res. 1993 May;299(3-4):147–156. [PubMed]
  • Feig DI, Sowers LC, Loeb LA. Reverse chemical mutagenesis: identification of the mutagenic lesions resulting from reactive oxygen species-mediated damage to DNA. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6609–6613. [PMC free article] [PubMed]
  • Frenkel K, Cummings A, Solomon J, Cadet J, Steinberg JJ, Teebor GW. Quantitative determination of the 5-(hydroxymethyl)uracil moiety in the DNA of gamma-irradiated cells. Biochemistry. 1985 Aug 13;24(17):4527–4533. [PubMed]
  • Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. [PubMed]
  • Harley CB, Reynolds RP. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. [PMC free article] [PubMed]
  • Hatahet Z, Kow YW, Purmal AA, Cunningham RP, Wallace SS. New substrates for old enzymes. 5-Hydroxy-2'-deoxycytidine and 5-hydroxy-2'-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2'-deoxyuridine is a substrate for uracil DNA N-glycosylase. J Biol Chem. 1994 Jul 22;269(29):18814–18820. [PubMed]
  • Hayes RC, LeClerc JE. Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I. Nucleic Acids Res. 1986 Jan 24;14(2):1045–1061. [PMC free article] [PubMed]
  • Hayes RC, Petrullo LA, Huang HM, Wallace SS, LeClerc JE. Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions. J Mol Biol. 1988 May 20;201(2):239–246. [PubMed]
  • Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, Benkovic SJ, Lerner RA. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science. 1989 Dec 8;246(4935):1275–1281. [PubMed]
  • Ide H, Kow YW, Wallace SS. Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro. Nucleic Acids Res. 1985 Nov 25;13(22):8035–8052. [PMC free article] [PubMed]
  • Ide H, Petrullo LA, Hatahet Z, Wallace SS. Processing of DNA base damage by DNA polymerases. Dihydrothymine and beta-ureidoisobutyric acid as models for instructive and noninstructive lesions. J Biol Chem. 1991 Jan 25;266(3):1469–1477. [PubMed]
  • Kohara Y, Akiyama K, Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. [PubMed]
  • Konigsberg W, Godson GN. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. [PMC free article] [PubMed]
  • Kow YW, Wallace SS. Mechanism of action of Escherichia coli endonuclease III. Biochemistry. 1987 Dec 15;26(25):8200–8206. [PubMed]
  • Kow YW, Faundez G, Melamede RJ, Wallace SS. Processing of model single-strand breaks in phi X-174 RF transfecting DNA by Escherichia coli. Radiat Res. 1991 Jun;126(3):357–366. [PubMed]
  • Kowalski D. A procedure for the quantitation of relaxed closed circular DNA in the presence of superhelical DNA: an improved fluorometric assay for nicking-closing enzyme. Anal Biochem. 1979 Mar;93(2):346–354. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Laspia MF, Wallace SS. Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli. J Bacteriol. 1988 Aug;170(8):3359–3366. [PMC free article] [PubMed]
  • Maccabee M, Evans JS, Glackin MP, Hatahet Z, Wallace SS. Pyrimidine ring fragmentation products. Effects of lesion structure and sequence context on mutagenesis. J Mol Biol. 1994 Feb 18;236(2):514–530. [PubMed]
  • Melamede RJ, Hatahet Z, Kow YW, Ide H, Wallace SS. Isolation and characterization of endonuclease VIII from Escherichia coli. Biochemistry. 1994 Feb 8;33(5):1255–1264. [PubMed]
  • Michaels ML, Cruz C, Grollman AP, Miller JH. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7022–7025. [PMC free article] [PubMed]
  • Michaels ML, Pham L, Cruz C, Miller JH. MutM, a protein that prevents G.C----T.A transversions, is formamidopyrimidine-DNA glycosylase. Nucleic Acids Res. 1991 Jul 11;19(13):3629–3632. [PMC free article] [PubMed]
  • O'Connor TR, Graves RJ, de Murcia G, Castaing B, Laval J. Fpg protein of Escherichia coli is a zinc finger protein whose cysteine residues have a structural and/or functional role. J Biol Chem. 1993 Apr 25;268(12):9063–9070. [PubMed]
  • Oshima T, Aiba H, Baba T, Fujita K, Hayashi K, Honjo A, Ikemoto K, Inada T, Itoh T, Kajihara M, et al. A 718-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 12.7-28.0 min region on the linkage map. DNA Res. 1996 Jun 30;3(3):137–155. [PubMed]
  • Parmley SF, Smith GP. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene. 1988 Dec 20;73(2):305–318. [PubMed]
  • Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A. 1975 Mar;72(3):784–788. [PMC free article] [PubMed]
  • Purmal AA, Kow YW, Wallace SS. Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro. Nucleic Acids Res. 1994 Jan 11;22(1):72–78. [PMC free article] [PubMed]
  • Radman M. An endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet-irradiated DNA. J Biol Chem. 1976 Mar 10;251(5):1438–1445. [PubMed]
  • Riek RP, Handschumacher MD, Sung SS, Tan M, Glynias MJ, Schluchter MD, Novotny J, Graham RM. Evolutionary conservation of both the hydrophilic and hydrophobic nature of transmembrane residues. J Theor Biol. 1995 Feb 7;172(3):245–258. [PubMed]
  • Rouet P, Essigmann JM. Possible role for thymine glycol in the selective inhibition of DNA synthesis on oxidized DNA templates. Cancer Res. 1985 Dec;45(12 Pt 1):6113–6118. [PubMed]
  • Russell CB, Thaler DS, Dahlquist FW. Chromosomal transformation of Escherichia coli recD strains with linearized plasmids. J Bacteriol. 1989 May;171(5):2609–2613. [PMC free article] [PubMed]
  • Sastry L, Alting-Mees M, Huse WD, Short JM, Sorge JA, Hay BN, Janda KD, Benkovic SJ, Lerner RA. Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5728–5732. [PMC free article] [PubMed]
  • Sharp PM, Li WH. Codon usage in regulatory genes in Escherichia coli does not reflect selection for 'rare' codons. Nucleic Acids Res. 1986 Oct 10;14(19):7737–7749. [PMC free article] [PubMed]
  • Shine J, Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. [PubMed]
  • Strniste GF, Wallace SS. Endonucleolytic incision of x-irradiated deoxyribonucleic acid by extracts of Escherichia coli. Proc Natl Acad Sci U S A. 1975 Jun;72(6):1997–2001. [PMC free article] [PubMed]
  • Tchou J, Kasai H, Shibutani S, Chung MH, Laval J, Grollman AP, Nishimura S. 8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4690–4694. [PMC free article] [PubMed]
  • Tchou J, Michaels ML, Miller JH, Grollman AP. Function of the zinc finger in Escherichia coli Fpg protein. J Biol Chem. 1993 Dec 15;268(35):26738–26744. [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Wagner JR, Hu CC, Ames BN. Endogenous oxidative damage of deoxycytidine in DNA. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3380–3384. [PMC free article] [PubMed]
  • Yacoub A, Augeri L, Kelley MR, Doetsch PW, Deutsch WA. A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities. EMBO J. 1996 May 1;15(9):2306–2312. [PMC free article] [PubMed]
  • Zhou M, Hatahet Z. An improved ligase-free method for directional subcloning of PCR amplified DNA. Nucleic Acids Res. 1995 Mar 25;23(6):1089–1090. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...